Copper is an essential micronutrient because of its role in enzymes that catalyze redox reactions. In humans, acquired copper-deficiency is rare because of the low daily requirement of copper, but copper-deficiency can accompany malnutrition, be associated with genetic disorders like Menkes syndrome or result from over- consumption of zinc supplements. And marginal copper deficiency, a contributing factor in cardiovascular disease, may not be uncommon. The long-term objective of this project is to i) discover the adaptive biochemical changes in copper-deficient cells in the context of specific cuproenzyme-containing pathways, ii) dissect the underlying regulatory mechanisms for achieving these changes, and iii) understand the physiological rationale for the adaptive modifications. Chlamydomonas reinhardtii is the model organism for the study because the well-defined synthetic growth medium facilitates studies of trace element homeostasis, and the full power of classical biochemical and genetic approaches can be combined with state of the art genomic and proteomic methods. The previous studies of copper-deficiency responses in Chlamydomonas established the concept of "back up" copper-independent enzymes that are expressed in -Cu cells to compensate for loss of function of cuproenzymes (e.g. cyt Cf,as a back up for plastocyanin and Crd2 as a back up for the multicopper ferroxidase), and led to the discovery of Crrl, a novel DNA binding protein that is required for all known copper-deficiency responses and is involved also in maintaining copper homeostasis in a copper replete cell. In this project period, the investigators will: 1) identify CRD2, which is required for high affinity iron uptake in a copper-deficient cell, determine its biochemical function in relation to Foxl (ferroxidase) and Ftrl (iron permease) function, and monitor its expression and sub-cellular location in response to copper and iron nutrition; 2) dissect 3 domains in the copper regulator Crrl - the DNA-binding SBP domain, ankyrin repeats and the Cys-rich C-terminus - by mutagenesis and in vivo and in vitro functional analysis including CuRE binding, metal binding and redox state, and determine the pattern of copper-dependent Crrl regulation and location, to develop a model for its action as a regulator of copper nutrition and hypoxia; and 3) identify novel targets of copper-deficiency by specialized oligonucleotide microarray analyses of -Cu vs. +Cu cells and errI vs. wild-type cells, in parallel with sub-proteomic gel- and multi-dimensional chromatography-based analyses of copper-replete vs. copper-deficient cells.